Concurrent presence of high serum uric acid and inflammation is associated with increased incidence of type 2 diabetes mellitus in Korean adult population

Although serum uric acid level and systemic inflammation have been highlighted as risk factors for type 2 diabetes mellitus (T2DM), little is known about these associations in the Korean population. Thus, we examined the individual and combined associations of serum uric acid and systemic inflammation (evaluated using high-sensitivity C-reactive protein [hs-CRP] measurement) with the future risk of T2DM. A total of 4152 Korean adults aged 45–76 years without T2DM, cancer, or gout at baseline in 2007–2008 from the Korean Genome and Epidemiology Study were followed up until 2016. Cox proportional hazard models were used to estimate the multivariable-adjusted hazard ratios (HRs) and 95% confidence intervals (CIs) of T2DM according to sex-specific tertiles of serum uric acid and hs-CRP levels after adjustment for confounders. During the mean follow-up of 7.3 years, 548 participants developed T2DM. High serum uric acid and hs-CRP levels were independently associated with an increased incidence of T2DM. The multivariable-adjusted HRs (95% CIs) for the incidence of T2DM in the highest tertiles of serum uric acid and hs-CRP were 1.54 (1.24–1.93) and 1.90 (1.48–2.43), respectively. High levels of serum uric acid and hs-CRP in combination were associated with an increased incidence of T2DM (HR: 4.69; 95% CI: 2.81–7.84) compared to low levels of serum uric acid and hs-CRP. These findings suggest that the combination of high serum uric acid and hs-CRP levels was significantly associated with an elevated incidence of T2DM; however, their synergistic effects were not observed in middle-aged and elderly Korean adults.

www.nature.com/scientificreports/ inflammatory pathways 14 . In addition, inflammatory cytokines increase the expression and activity of xanthine oxidase, an enzyme that catalyzes the conversion of uric acid precursors into uric acid 15 . Therefore, we investigated the independent and interactive effects of serum uric acid level and inflammation on metabolic dysfunction. Although a few studies have evaluated the contributory effects of uric acid and inflammation on metabolic disorders, they were limited to cross-sectional and longitudinal studies with small sample sizes or short follow-up periods. Additional evidence based on prospective cohort studies with longer follow-up periods are needed to determine whether serum uric acid or inflammation is prospectively associated with T2DM risk and whether the concurrent presence of these two factors interacts synergistically to develop T2DM. This study aimed to explore the independent role of uric acid and systemic inflammation (measured by highsensitivity CRP [hs-CRP] levels) in predicting the future risk of T2DM. We also prospectively investigated the combined effects of serum uric acid and hs-CRP levels on the incidence of T2DM in a population-based cohort of middle-aged and elderly Korean adults.

Results
Baseline characteristics. During a mean follow-up period of 7.3 ± 1.6 years (30,365 total person-years), 548 new cases of T2DM were identified. The main characteristics of the study participants at baseline across the sex-specific tertiles of serum uric acid and hs-CRP levels are summarized in Table 1. Area of residence, education level, regular physical activity, body mass index (BMI), waist circumference (WC), diastolic blood pressure, triglycerides, total cholesterol, high-density lipoprotein (HDL)-cholesterol, and fasting blood glucose at baseline significantly differed according to tertiles of serum uric acid and hs-CRP levels (all, P < 0.05). Participants in the highest tertile of serum uric acid levels (serum uric acid ranges 6.2 to 11.8 mg/dL for men; 4.5 to 10.6 mg/ dL for women) were more likely to live in Ansung (rural area), drink more alcohol, exercise regularly, and have high BMI, WC, diastolic blood pressure, triglycerides, total cholesterol and fasting blood glucose levels and low HDL-cholesterol levels. Participants with the highest tertile of hs-CRP levels (hs-CRP ranges 1.05 to 9.82 mg/L for men; 0.90 to 9.88 mg/L for women) tended to be older, urban area (Ansan) residents, less educated, current smokers, less physically active, and have high BMI, WC, systolic and diastolic blood pressure, triglycerides, total cholesterol, and fasting blood glucose levels. Those from the highest tertile of serum uric acid or hs-CRP levels were more likely to have low HDL-cholesterol levels.
Independent associations of serum uric acid and hs-CRP on incident T2DM risk. The incidence rates, hazard ratios (HRs) and 95% confidence intervals (CIs) for T2DM are provided according to tertiles, continuous measures, and dichotomous categories of serum uric acid and hs-CRP levels in Tables 2 and 3. Participants in the highest tertile of serum uric acid had a 54% increased risk of T2DM compared to those in the lowest tertile after controlling for potential confounders (fully adjusted HR: 1.54; 95% CI: 1.24-1.93; P trend < 0.0001) ( Table 2). Moreover, the fully adjusted HR was 1.47 (95% CI: 1.02-2.12) per 1 log mg/dL increase in serum uric acid levels. However, when we dichotomized serum uric acid levels (hyperuricemia vs. non-hyperuricemia), the associations were not significant (fully adjusted HR: 1.12; 95% CI: 0.87-1.44).
Combined associations of serum uric acid and hs-CRP on incident T2DM. The associations between serum uric acid and hs-CRP levels and the incidence of T2DM are shown in Table 4. Although the test for the multiplicative interaction between serum uric acid and hs-CRP levels was not statistically significant (P interaction = 0.3790), when cross-classifying participants by both exposure variables, the risk of developing T2DM was the highest among participants with the combination of the highest tertile of serum uric acid and hs-CRP levels (HR: 4.69; 95% CI: 2.81-7.84) compared with the opposite extreme.
Predicting incident T2DM with serum uric acid and hs-CRP. A comparison of the performance of each model in predicting T2DM is presented in Table 5. The model with both serum uric acid and hs-CRP levels showed the best predictive accuracy, with an area under the receiver operating characteristic curve (AUC) between 0.731 and 0.930, and the highest Uno's C-statistic (0.758) during the follow-up periods. C-statistics of the prediction models including at least one of the markers of serum uric acid and hs-CRP were significantly higher than those of the conventional model (all, P < 0.0001). The predictive model including both serum uric acid and hs-CRP levels provided a better C-statistic than the model with only serum uric acid levels (P = 0.0030); however, there was no significant difference in the model with only hs-CRP levels (P = 0.2445).

Discussion
Our prospective cohort study of middle-aged and elderly Korean adults (mean follow-up time of 7.3 years) showed that elevated serum uric acid and hs-CRP levels increased the risk of T2DM development. For each 1 log unit increase in serum uric acid (mg/dL) and hs-CRP (mg/L), the incidence of T2DM increased by 47% and 17%, respectively. These associations were independent of conventional T2DM risk factors such as sex, age, residential area, education level, smoking status, alcohol consumption, physical activity, BMI, WC, blood pressure, serum lipid and fasting blood glucose levels, and family history of diabetes. Furthermore, high levels of www.nature.com/scientificreports/ serum uric acid, in combination with hs-CRP, exacerbated the future risk of developing T2DM, but these two factors did not act synergistically.
To the best of our knowledge, this is the first population-based cohort study to investigate the combined effects of serum uric acid and hs-CRP levels on the incidence of T2DM from a longitudinal perspective. Previous studies conducted in the United States and Europe have demonstrated positive associations between serum uric acid levels and the risk of T2DM. Data from the Framingham Heart Study 9 and Rotterdam Study 8 showed that elevated serum uric acid levels were significantly associated with an increased risk of T2DM. However, the role of serum uric acid level in T2DM development in Asian populations has not been consistent. A prospective study of Japanese men reported no association between serum uric acid levels and the incidence of T2DM 16 . In contrast, a cohort study of 2,690 Chinese adults aged 35-97 years showed that the risk of T2DM was higher in individuals with the highest levels of serum uric acid than in those with the lowest levels (relative risk for Q5 Table 1. Baseline characteristics of the study participants by tertile of serum uric acid and high-sensitivity C-reactive protein levels in Korean adults aged 45-76 years. T tertile; hs-CRP high-sensitivity C-reactive protein; HDL-cholesterol high-density lipoprotein cholesterol. 1 Cut-offs for tertiles 1-3 of serum uric acid levels are as follows: < 5.2, 5.2-6.1, and > 6.1 mg/dL in men and < 3.8, 3.8-4.4, and > 4.4 mg/dL in women, respectively. 2 Cut-offs for tertiles 1-3 of hs-CRP levels are as follows: < 0.45, 0.45-1.04, and > 1.04 mg/L in men and < 0.38, 0.38-0.89, and > 0.89 mg/L in women, respectively. 3 Values are number (percentage) for categorical variables and mean ± standard deviation for continuous variables.  17 . Along with a previous Chinese study, the present study found significant positive associations between serum uric acid levels and the incidence of T2DM, regardless of how we defined the exposure variable in the analytic models. Our findings reflect the robustness of these associations and suggest the potential role of elevated serum uric acid levels as an independent risk factor for T2DM among Korean adults.
As insulin resistance and T2DM may result from subclinical chronic low-grade inflammation, inflammatory cytokine levels have been reported to serve as effective biomarkers for T2DM 18 . CRP is commonly used as a marker of systemic inflammation, and its association with T2DM has been evaluated in various non-diabetic populations. Some cross-sectional studies have shown that CRP levels vary substantially among different ethnic groups 6,19 . Kelley et al. 6 compared CRP levels among different ethnicities and reported that CRP concentrations were the highest among African Americans (median: 3.2 mg/L), followed by Hispanics (median: 2.3 mg/L), Caucasians (median: 1.5 mg/L), Chinese (median: 0.7 mg/L), and Japanese (median: 0.5 mg/L). In our study, the median hs-CRP level in Koreans was 0.6 mg/L, which was similar to than in other Asian groups and much lower than that in Westerners. Although differences in CRP distributions across populations have been acknowledged, Table 2. Adjusted hazard ratios (with 95% confidence intervals) for type 2 diabetes according to serum uric acid levels in Korean adults aged 45-76 years. T tertile; HR hazard ratio; CI confidence interval. 1 Cut-offs for tertiles 1-3 of serum uric acid levels are as follows: < 5.2, 5.2-6.1, and > 6.1 mg/dL in men and < 3.8, 3.8-4.4, and > 4.4 mg/dL in women, respectively. 2 Hyperuricemia is defined as serum uric acid levels ≥ 7 mg/dL in men and ≥ 6 mg/dL in women. 3 Model 1: adjusted for sex (men or women) and age (years); Model 2: additionally adjusted for area of residence (Ansan or Ansung), education level (≤ elementary school, middle/high school, or ≥ college), smoking status (never, past, or current), alcohol consumption (g/d), regular physical activity (yes or no), body mass index (kg/m 2 ), waist circumference (cm), systolic and diastolic blood pressure (mmHg), triglyceride (mg/dL), total cholesterol (mg/dL), high-density lipoprotein cholesterol (mg/dL) and fasting blood glucose level (mg/dL), and family history of diabetes (yes or no); Model 3: additionally adjusted for logtransformed hs-CRP levels.  www.nature.com/scientificreports/ previous analyses of cohort studies from various populations indicated relatively consistent findings, suggesting a positive association between CRP levels and the incidence of T2DM. A recent meta-analysis of 19 prospective studies conducted predominantly in Western countries showed a 25% increase in the risk of T2DM per 1 log pg/ mL increment in CRP concentrations 5 . In agreement with our results, previous studies with Asian populations, in which relatively low CRP levels were observed, also reported positive associations between CRP levels and the risk of T2DM development 20,21 . Moreover, although we presented the prospective associations of hs-CRP levels with an increased risk of T2DM among Korean adults, it is limited to determining Korean-specific CRP cut-off values for predicting the future risk of T2DM. Therefore, further studies are needed to establish the optimal hs-CRP cut-off points that could identify high-risk groups for T2DM development among Koreans. Several studies have shown a significant relationship between serum uric acid concentrations and the levels of CRP and pro-inflammatory markers involved in the causal pathways of T2DM. In observational studies, serum uric acid levels were positively associated with CRP levels 22,23 , TNF-α 23 , and IL-6 13 . However, the underlying mechanisms by which uric acid increases the levels of inflammatory markers remain unclear. One possible explanation is that damaged cells and tissues release uric acid, which triggers inflammatory cytokines 24 . Results from an experimental study with human vascular and endothelial cells support the hypothesis that high serum uric acid concentrations stimulate CRP expression 11 . In contrast, pro-inflammatory cytokines play an important role in uric acid metabolism by upregulating the expression of xanthine oxidase 14 , which plays a key role in uric acid metabolism by converting xanthine to uric acid 25 . The increased expression of xanthine oxidase promotes the production of uric acid and superoxide free radicals 15 , resulting in endothelial dysfunction that may cause T2DM 12 . Therefore, studies exploring the role of the vicious cycle between uric acid and inflammation in the onset of metabolic disorders, including T2DM, are needed. However, to date, although the separate effects of serum uric acid and hs-CRP levels on the risk of T2DM have been recognized 26,27 , the combined effect of these two biomarkers on T2DM has not been investigated. Only a few cross-sectional studies have shown that the concurrent presence of elevated uric acid and CRP levels is associated with the risk of hypertension 28 , metabolic Table 4. Association of type 2 diabetes by serum uric acid and high-sensitivity C-reactive protein level strata in Korean adults aged 45-76 years. T tertile; hs-CRP high-sensitivity C-reactive protein; HR hazard ratio; CI confidence interval. 1 Cut-offs for tertiles 1-3 of serum uric acid levels are as follows: < 5.2, 5.2-6.1, and > 6.1 mg/dL in men and < 3.8, 3.8-4.4, and > 4.4 mg/dL in women, respectively. 2 Cut-offs for tertiles 1-3 of hs-CRP levels are as follows: < 0.45, 0.45-1.04, and > 1.04 mg/L in men and < 0.38, 0.38-0.89, and > 0.89 mg/L in women, respectively. 3 Individuals in the lowest tertile of serum uric acid and hs-CRP levels are used as the reference group. The model is adjusted for sex (men or women), age (years), area of residence (Ansan or Ansung), education level (≤ elementary school, middle/high school, or ≥ college), smoking status (never, past, or current), alcohol consumption (g/d), regular physical activity (yes or no), body mass index (kg/m 2 ), waist circumference (cm), systolic and diastolic blood pressure (mmHg), triglyceride (mg/dL), total cholesterol (mg/ dL), high-density lipoprotein cholesterol (mg/dL) and fasting blood glucose level (mg/dL), and family history of diabetes (yes or no). ** P < 0.001.  Table 5. Overall ability of models predicting type 2 diabetes incidence in Korean adults aged 45-76 years.
AUC area under the receiver operating characteristic curve; hs-CRP high-sensitivity C-reactive protein; T2DM, type 2 diabetes mellitus. 1 The conventional model is adjusted for sex (men or women), age (years), area of residence (Ansan or Ansung), education level (≤ elementary school, middle/high school, or ≥ college), smoking status (never, past, or current), alcohol consumption (g/day), regular physical activity (yes or no), body mass index (kg/m 2 ), waist circumference (cm), systolic and diastolic blood pressure (mmHg), triglyceride (mg/dL), total cholesterol (mg/dL), high-density lipoprotein cholesterol (mg/dL) and fasting blood glucose level (mg/ dL), and family history of diabetes (yes or no). 2 Uno's C-statistic from Cox linear models. 3 Compared with C-statistic of conventional model. 4 Compared with C-statistic of + serum uric acid model. 5 Compared with C-statistic of + hs-CRP model. www.nature.com/scientificreports/ syndrome, and insulin resistance 29 . In our study, findings from the large cohort with long-term follow-up may provide new insights into the prospective relationships of coexisting elevated serum uric acid and hs-CRP levels with the incidence of T2DM, even though their effects were not synergistic. In addition, we estimated the C-statistics and AUCs to evaluate the usefulness of serum uric acid with concomitant hs-CRP levels in predicting the future risk of T2DM. Our findings imply that identifying concurrent elevated serum uric acid and hs-CRP levels as T2DM risk factors may expand opportunities for the early detection and prevention of T2DM. The strengths of this study include its prospective design, relatively long follow-up period, and the inclusion of detailed information on potential confounders. Furthermore, this study extended the findings of previous studies conducted in Western populations and provided additional evidence of the separate and combined contributions of serum uric acid and hs-CRP levels to the incidence of T2DM among populations with relatively low hs-CRP levels. This study is not without potential limitations. First, the study population was composed of middle-aged and older adults residing in specific regions of Korea, which may limit the generalizability of the findings to the entire Korean population. However, the distributions of baseline serum uric acid and hs-CRP levels in our study population did not vary significantly from those of previous nationwide or cohort studies 30,31 ; Thus, the associations observed in this study might be similar for other Korean populations. Second, dietary data were not available at baseline. However, we could evaluate the effect of alcohol consumption, which is a major dietary factor affecting serum uric acid levels, in the analytical models. Moreover, even after adjusting the analytical models for confounders related to T2DM, significant associations between serum uric acid and hs-CRP levels and the incidence of T2DM were still maintained. Third, all analyses were based on single measurements of serum uric acid and hs-CRP levels, which limited the conclusions regarding the effects of changes in serum uric acid and inflammation levels on T2DM development over time. Future longitudinal studies that include repeated measurements of serum uric acid and hs-CRP levels and incorporate dietary information are warranted.

Conclusions
In summary, elevated levels of serum uric acid and hs-CRP were independently associated with an increased incidence of T2DM. Although there was no significant synergistic effect of these two indicators, the concurrent presence of elevated serum uric acid and hs-CRP levels may exacerbate the risk of T2DM development in a large Korean cohort aged 45 years and older. Our findings suggest that monitoring serum uric acid and hs-CRP levels can be an effective strategy for identifying individuals at high risk of T2DM development among Korean adults.
Research design and methods. Data source and study population. We used data from the Ansan-Ansung cohort study of the Korean Genome and Epidemiology Study (KoGES). In two Korean cities (Ansan and Ansung), 10,030 Korean adults aged between 40 and 69 years were recruited between 2001 and 2002 and followed up biennially. As previously described, data on sociodemographics, lifestyle, medical and medication history, and reproductive health were collected. Health examinations and blood and urine tests were performed by trained staff and interviewers using standardized protocols at baseline and subsequent follow-up examinations 32 . In 2018, the Korea Center for Disease Control and Prevention (KCDC) newly released data on the levels of 15 biomarkers, including serum uric acid, from stored serum samples collected at the 3 rd follow-up examination between 2007 and 2008. Therefore, in this study, data from the 3rd follow-up examination were considered as the baseline to include data on serum uric acid levels. From a total of 6688 participants who completed the 3 rd follow-up examination (2007-2008), 1,110 were excluded because stored serum samples were not available. We further excluded participants with T2DM (n = 1252), any type of cancer (n = 64), or gout (n = 6). Of these, 37 participants were not suitable for the current study because of missing information on covariates. To rule out confounding by acute infection, participants with hs-CRP levels of > 10 mg/L were excluded from the final analysis (n = 67). Finally, 4152 Korean adults (1875 men and 2277 women) who were followed-up until 2016 were included in this study (Fig. 1). The KoGES study was reviewed and approved by the Institutional Review Board of the Korea Centers for Disease Control and Prevention, and written informed consent was obtained from all the participants. All methods were performed in compliance with relevant institutional guidelines and regulations. The protocol was reviewed and approved by the Institutional Review Board of Korea National University of Education on November 24, 2020 (IRB No. KNUE-202011-BMBR-0102-01).
Assessment of serum uric acid and hs-CRP. Blood samples for the laboratory tests were collected after overnight fasting. Serum uric acid and hs-CRP concentrations were assessed using enzymatic colorimetric methods with an automatic analyzer (ADVIA 1650 and 1800, Siemens, Tarrytown, NY, USA). Serum uric acid and hs-CRP levels, the main exposure variables of interest, were analyzed as tertiles based on sex-specific distributions and as continuous measures. Both exposure variables were analyzed as dichotomous variables. For dichotomous analyses, hyperuricemia was defined as serum uric acid levels ≥ 7 mg/dL in men and ≥ 6 mg/dL in women 33 . This is the saturation point of monosodium urate, which is the prevalent form of uric acid in extracellular fluids 34 . High inflammation was defined as hs-CRP levels > 2 mg/L in both men and women, as previously used in the Asian population 35 .
Ascertainment of T2DM. The incidence of T2DM was determined if participants had fasting blood glucose levels ≥ 126 mg/dL or 2-h oral glucose tolerance tests (OGTT) ≥ 200 mg/dL at follow-up examinations, based on the World Health Organization 36 and American Diabetes Association 37 . In addition, participants who were newly diagnosed with T2DM after the previous examination or who used insulin or oral anti-diabetic medicine were considered T2DM cases. www.nature.com/scientificreports/ Statistical analyses. Differences in the general characteristics of study participants across the categories of serum uric acid and hs-CRP levels were tested using the Mantel-Haenszel chi-square test for categorical variables and generalized linear regression for continuous variables, respectively. Data for serum uric acid and hs-CRP levels were right-skewed and log-transformed before the analysis. Multivariable Cox proportional hazard models were used to estimate the future risk of T2DM development. To test for linear trends, we used the median of each tertile of serum uric acid and hs-CRP levels as continuous variables. The following covariates were used for adjustment: sex (men or women) and age (years) were adjusted in Model 1; area of residence (Ansan or Ansung), education level (≤ elementary school, middle/high school, or ≥ college), smoking status (never, past, or current), alcohol consumption (g/d), regular physical activity (yes or no), BMI (kg/m 2 ), WC (cm), systolic and diastolic blood pressure (mmHg), triglyceride (mg/dL), total cholesterol (mg/dL), HDL-cholesterol (mg/dL), fasting blood glucose (mg/dL), and family history of diabetes (yes or no) were additionally adjusted in Model 2; log-transformed serum uric acid or hs-CRP levels were additionally adjusted in Model 3. To explore the combined effect of serum uric acid and hs-CRP levels on the incidence of T2DM, we cross-classified participants according to tertiles of serum uric acid and hs-CRP levels and analyzed the interactions between serum uric acid and hs-CRP levels and the incidence of T2DM. To test for potential interactions between serum uric acid and hs-CRP levels, we applied multiplicative interaction by including corresponding interaction terms (serum uric acid level × serum hs-CRP level, treated as log-transformed continuous variables) in the fully adjusted models. The lowest tertile of both variables was used as the reference.
Uno's C-statistics from the Cox models were used to assess the differences in the overall prediction ability among the T2DM incidence predictive models. The conventional prediction model included established T2DM risk factors such as sex, age, residential area, education level, smoking status, alcohol consumption, regular www.nature.com/scientificreports/ physical activity, BMI, WC, systolic and diastolic blood pressure, triglyceride, total cholesterol, HDL-cholesterol, fasting glucose levels, and family history of diabetes. In the other three prediction models, serum uric acid and hs-CRP levels were additionally included individually or together, and the predictive abilities of the three models were compared with the conventional model. SAS version 9.4 (SAS Institute, Cary, NC, USA) was used for all statistical analyses. Statistical tests were twosided, and a P value < 0.05 was considered significant.